Acellular biologic composition and method of manufacture

ABSTRACT

A biological composition has a mixture of mechanically selected allogeneic biologic material derived from bone marrow. The mixture has non-whole cellular components including vesicular components and active and inactive components of biological activity, cell fragments, cellular excretions, cellular derivatives, and extracellular components. The mixture including non-whole cell fractions including one or more of exosomes, transcriptosomes, proteasomes, membrane rafts, lipid rafts. The mixture is compatible with biologic function.

RELATED APPLICATIONS

This application is a division of co-pending U.S. application Ser. No.14/682,523 filed on Apr. 9, 2015 entitled “Acellular BiologicComposition And Method Of Manufacture”.

TECHNICAL FIELD

This invention is a tissue regenerative biological composition. Morespecifically, a composition at least in part formed from bone marrow anda method of manufacture and use of said composition with an acellularmixture.

BACKGROUND OF THE INVENTION

In the area of tissue regeneration or repair, the use of stem celltherapy has been widely touted.

Often, these inventions describe isolating the stem cells, purifying andculturally expanding mesenchymal stem cells. In U.S. Pat. No. 5,837,539,entitled “Monoclonal Antibodies For Human Mesenchymal Stem Cells”,Arnold Caplan et al. reported that the cells are preferably culturallyexpanded, but suggest it is possible to use the stem cells withoutculture expansion. Caplan also describes a way to isolate stem cells.

A major technological hurdle to producing a safe allogeneic compositionwith viable cells has been the need to approach a fraction of riskapproaching zero by removing all antigenic properties that lead toinflammation factors in a separation to yield only a certain stromalcell type. This has proven both difficult and degrading the quantity ofviable cells that can be effectively harvested.

The present invention has yielded a biological composition that is safeand achieves and does so in a method that allows the resultant mixtureto be recovered from bone marrow wherein the mixture unexpectedlyexhibits evidence of viability independent of mesenchymal cells in thedose and sustains a legacy or memory of the lineages from where theacellular biological composition came which retain the ability tosupport the emergence of new tissue forms including bone and othertissues.

These and other benefits of the present invention and the method ofpreparing it are described hereinafter.

SUMMARY OF THE INVENTION

A biological composition has a mixture of mechanically selectedallogeneic biologic material derived from bone marrow. The mixture hasnon-whole cellular components including vesicular components and activeand inactive components of biological activity, cell fragments, cellularexcretions, cellular derivatives, and extracellular components. Themixture including non-whole cell fractions including one or more ofexosomes, transcriptosomes, proteasomes, membrane rafts, lipid rafts.The mixture is compatible with biologic function.

The mixture of mechanically selected material derived from bone marrow.The biological composition preferably has bone particles. The boneparticles can be added to the mixture derived from bone marrow. The boneparticles include a mixture of cortical bone particles and cancellousbone particles.

The combination of non-whole cell components with a select number ofnon-whole cell fractions sustains pluripotency in the cells. In apreferred embodiment, the biological composition is predisposed todemonstrate or support elaboration of active volume or spatial geometryconsistent in morphology with that of endogenous bone. The biologicalcomposition extends regenerative resonance that compliments or mimicstissue complexity. The mixture is treated in a protectant orcryoprotectant prior to preservation or cryopreservation or freezedrying. The composition can be maintained at ambient temperature priorto freeze drying. The protectant or cryoprotectant creates a physical orelectrical or chemical gradient or combination thereof for tissueregeneration. The gradient can have a physical characteristic of modulusor topography, such as charge density, field shape or cryo or chemotoxic tendencies. The gradient can have a chemical characteristic ofspatially changing compositions of density or species of functionalmolecules, wherein the molecules can offer a fixed catalytic function asa co-factor. Also, the gradient can have an electrical characteristic ofcharge based or pH based or electron affinities that confermetastability in biologic potential.

The bone marrow mixture which is derived from a cadaver hasseparation-enhanced non-whole cell fractions vitality including one ormore of the following: separating the fractions from cells heightenstheir vitality, reversing “arrest” of donors, responsive molecularcoupling, matrix quest in neutralizing inflammation or satience bybalancing stimulus for repair. The protectant or cryoprotectant is apolyampholyte. The regenerative resonance occurs in the presence orabsence of a refractory response. When using a cryoprotectant, thecryopreservation occurs at a temperature that is sub-freezing whereinthe cryopreservation temperature is from 0 degrees C. to −200 degrees C.The protection may also be achieved by non-cryogenic means.

The biological composition's non-whole cellular component also caninclude organelle fragments and the active and inactive components ofbiological activity which can also include extants of the humanmetabolome.

A method of making a biological composition of the present invention hasthe steps of: collecting, recovering and processing bone marrow from acadaver donor; mechanically separating the cellular or non-cellularcomponents or a combination thereof of bone marrow from cadaverous bone;concentrating by centrifugation and filtering; separation by densitygradient centrifugation; collecting non-cellular fractions ornon-cellular components or a combination thereof of predetermineddensity; washing the non-whole cellular fractions or non-cellularcomponents or a combination thereof to create the mixture; quantifyingconcentrations of non-cellular fractions components at a non-zeroentity; suspending to a predetermined concentration in a polyampholytecryoprotectant; freezing the mixture at a predetermined controlled rate;and packaging a bone blend having particles in the size range of 100 to300 μm of demineralized cortical bone, mineralized cortical bone andmineralized cancellous bone either within the mixture or separate. Theseparticle size ranges can vary higher or lower depending on theapplication. At the time of use, the mixture is thawed by immersion in awarm water bath for 2-3 minutes at 37 degrees C. It is diluted in salinewithout spinning; and then the diluted mixture, with or without the boneblend being intermixed, can be implanted by packing, injection,scaffolding or any other suitable means into a patient.

Definitions

DNase—deoxyribonuclease is any enzyme that catalyzes the hydrolyticcleavage of phosphodiester linkages in the DNA backbone, thus degradingDNA.

DMEM, DMEM/LG—Dulbecco's Modified Eagle Medium, low glucose. Sterile,with: Low Glucose (1 g/L), Sodium Pyruvate; without: L-glutamine, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)

DPBS—Dulbecco's Phosphate Buffered Saline.

CBT-MIXER—Mixing blade for Cancellous Bone Tumbler Jar.

Cold Media—Media used during the preparation of vertebral bodies forinitial processing.

Cryopreserved—Tissue frozen with the addition of, or in a solutioncontaining, a cryoprotectant agent such as glycerol, ordimethylsulfoxide, or carboxylated poly-1-lysine.

Freeze Dried/Lyophilized—Tissue dehydrated for storage by conversion ofthe water content of frozen tissue to a gaseous state under vacuum thatextracts moisture.

Normal Saline—0.9% Sodium Chloride Solution.

Packing Media—Media used during initial processing and storage of theprocessed vertebral bodies prior to bone decellularization.

PBS—Phosphate Buffered Saline.

Processing Media—Media used during bone decellularization that maycontain DMEM/Low Glucose no phenol red, Human Serum Albumin, Heparin,Gentamicin and DNAse.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. The invention will be described by way of exampleand with reference to the accompanying drawings in which:

FIG. 1 shows a photograph of a cut vertebral body taken from a spine ofa cadaver donor.

FIG. 2 shows a photograph of the vertebral body after being cut intocubic pieces and immersed in a packing media.

FIG. 3 shows a photograph of the bulk bone material after being groundand immersed in packing media and placed in a jar for later tumbling.

FIG. 4 shows a photograph of the jar with a CBT-Mixer connected to atumbler.

FIG. 5 is a photograph of an exemplary sieve device having sieves sizedto separate the solid material.

FIG. 6 shows a photograph of two 50 ml vials, the one on the left beingprior to centrifuging with the Ficoll that is commercially available atthe bottom and the material above it. The 50 ml vial on the right isafter centrifuging showing the non-whole cell fraction interface layer.

FIG. 7 is a photograph showing the four tumbling steps 1-4 by exemplarycollection and Ficoll separation of the decanted fluids, the fluid intumble 1 being completely discarded to remove unwanted debris.

FIG. 8 shows a photograph of two 50 ml vials, the one on the left beingprior to centrifuging with a sucrose gradient that is commerciallyavailable at the bottom and the material above it. The 50 ml vial on theright is after centrifuging showing the non-whole cell fraction abovethe interface layer.

FIG. 9 is a representative photograph of the final packaging.

FIG. 10 is a photograph showing the ground bone.

DETAILED DESCRIPTION OF THE INVENTION

With reference to the present invention which is a tissue regenerativebiological composition 100 made from bone marrow 200, it is believedbest understood by the methods used to process and recover thebiological composition, as illustrated in the FIGS. 1-6.

The first steps are to collect, recover and process bone marrow 200 froma cadaver donor. To do this, the spine is removed aseptically from thecadaver and the resultant spine segment is covered by cold media. Thecold media has 0.5 ml of Heparin; 10,000 units/ml per 500 ml of DMEM.DMEM is a sterile solution with low glucose (1 g/L), Sodium Pyruvate;without L-glutamine, or HEPES. This cold media is used for packaging thespine segments for later processing. At this point the spine segmentincludes a plurality of vertebral bodies 202. The clinical technicianmust remove as much soft tissue as possible and cut each vertebral body202 with a saw. These vertebral bodies 202, once cleaned, of alladherent soft tissue around the cortical surfaces will look as shown inFIG. 1.

Once a cleaned vertebral body 202 is obtained, the next step involvescutting each vertebral body 202 into pieces, each piece 204 roughly 1cm³. The cut pieces 204 being immersed in a packing media 400. Theexemplary packing media can be DMEM with 0.5 ml Heparin and 1.25 ml ofDNAse added.

Once all the vertebral bodies 202 have been cut, the pieces 204 aretaken to the bone grinder. The bone is ground into 4-10 mm pieces usingpacking media 400 to help the pieces go through the grinder. The groundbone 206 (bulk cortical-cancellous crushed) and all of the packing media400, estimated volume of 500 ml are transferred into a jar 300 where0.5-1.0 ml of Gentamicin is added to the jar 300 with ground bone 206and packing media 400. At this point, the crushed bone 206, includingcellular soft marrow 200, is intermixed.

The step of mechanically separating these cellular components of bonemarrow 200 from the cadaverous bone is next performed. Transferring thebulk cortical-cancellous bone chips into a new jar with a CBT-Mixer inthe jar. The bulk cortical-cancellous bone chips 206 will go throughfour cycles as summarized in the table below. Each cycle, after cycle 1,contains three steps using a bone tumbler 500 and sieve set 600. Thesieve set 600 has screens 602 of various sizes, for example 500 μm and180 μm, as shown in FIG. 5.

Step Cycle 1 Cycle 2 Cycle 3 Cycle 4 Bone 30 minutes. 30 minutes 30minutes 30 minutes Tumbler Using Using Using Using 500 mL 500 mL 500 mL400 mL Processing Processing Processing Processing Media Media MediaMedia Sieve Set Use the Use the Use the Use the 500-μm 500-μm, 500-μm,500-μm, and the 180-μm 180-μm 180-μm bottom and bottom and bottom andbottom pan sieve. pan sieve. pan sieve. pan sieve. Discard CollectCollect Collect decanted decanted decanted decanted fluid. fluid. fluid.fluid. Centrifuge N/A Use Use Use decanted decanted decanted fluid.fluid. fluid.

In cycle 1, the decanted fluid 210 is discarded. To best understandthis, an exemplary FIG. 7 shows conical tubes with the decanted fluidsafter each cycle followed by Ficoll separation. Tumble 1 or Cycle 1 hasmost of the unwanted cells and debris as evidenced by its dark and redappearance whereas each subsequent cycle 2, 3 and 4 are progressivelycleared. This FIG. 7 is only to illustrate the effects of multipletumbles 1-4 and the value in discarding the decanted liquid 210 afterthe first tumble 1.

After each subsequent sieving of the bulk bone material 206, thedecanted fluid 212, 214, 216 containing the mixture with whole cells iscollected and put into a collection jar. When the next three cycles arecomplete and the decanted fluid is all placed in the collection jarcomingling the fluids 212, 214 and 216 to form a decanted fluid 220.Then the centrifugation of the combined decanted fluid 220 occurs byplacing the fluid 220 in a number of 250 ml conical tubes using a 100 mlpipette. The centrifuge is programmed to 280×g for 10 minutes at roomtemperature, preferably about 20 degrees C. The fluid 220 is passedthrough a blood filter to further remove any bone or spicules or clumpsfrom the suspended cells. This completes the step of centrifuging andfiltering. At this point, the mixture including whole cells 240 has beenseparated from the soft marrow tissue 200 and the remaining cancellousand cortical bone is discarded.

After this as shown in FIG. 6, the step of separating the cells 240 fromthe non-whole cellular components by a density centrifugation occurs.The whole cells 240 are in the interface and the non-whole cellcomponents are in the supernatant above the interface. The mixtureincluding is placed in 50 ml conical tubes 20 with Ficoll 800 andundergoes a Ficoll-Paque separation under centrifugation wherein a celldensity gradient is established by spinning at 400×g for 30 minutes atroom temperature, preferably about 20 degrees C. The mixture includescellular or non-cellular components or a combination thereof. All fluid211 above the interface is removed include the desired non-whole cellcomponents which exclude the whole cells 240, 250.

Typically, non-whole cell fragments, or membrane components have adiameter of 40-100 nm and can be separated within a density of 1.13-1.19g/mL in a sucrose solution, and can be sedimented by centrifugation at100,000 g. In fact, these fragments, or cell fractions, ormicrovesicles, have been collectively referred to as exosomes. Rangingin size from 20-1000 nm in diameter, they have been similarly referredto as nanoparticles, microparticles, shedding microvesicles, apoptoticblebs, and human endogenous retroviral particles. There are few firmcriteria distinguishing one type of microvesicle from the other.

Following removal of the cell fraction, the supernatant is furtherfiltered through 0.45 and 0.2 μm filters. Exosomes are further collectedand separated within the suspension in multiple centrifugation stepswith increasing centrifugal strength to sequentially pellet cells (300g), microvesicles (10,000 g) and ultimately exosomes (100,000 g). Cellsare deliberately removed to achieve the non-whole cell fragments andmicrovesicles.

Subsequent separation using density gradient-based isolation, usingsucrose or commercially available prep can be applied to obtain morepure exosome preparations. Recent reports encouraging the use ofiodixanol-based gradients for improved separation of exosomes fromviruses and small apoptotic bodies are considerations left open to beadopted or adapted in refinement. Differing from sucrose, iodixanolforms iso-osmotic solutions at all densities, thus better preserving thesize of the vesicles in the gradient, and both technologies areavailable to best isolation technology. In addition to these traditionalisolation techniques, easy-to-use precipitation solutions, such asExoQuick™ and Total Exosome Isolation™ (TEI), that have beencommercialized reduce the need for expensive equipment or technicalknow-how. Although their mode-of-action has not been disclosed orvalidated, these kits are commonly used.

Once the mixture is completed, the method can include additional steps.This leads to the use of a bone blend 102 shown in FIG. 10, preferablyfrom the same vertebral bone or at least bone from the same donor.

When the mixture is prepared, it can have whole cells or even no wholecells, but will have the mechanically selected non-whole cellularcomponents including vesicular components and active and inactivecomponents of biological activity, cell fragments, cellular excretions,cellular derivatives, and extracellular components.

In one embodiment, the composition includes the whole cells in themixture. In that embodiment, it is possible to provide bone particleswith the mixture either in the mixture or separately to be combined atthe time of use.

In one embodiment, the bone is ground to a particle size of 100-300 μm,see FIG. 11. The bone mixture has 1.5 cc of mineralized cancellous bone104, 1.5 cc of mineralized cortical bone 105 and 2.0 cc of demineralizedcortical bone 106 yielding 30 percent, 30 percent and 40 percentrespectively of the total 5 cc (5 gram) of bone material 102. The rangescoincide with the 1 cc of mixture when resuspended in 3 cc of saline toprovide a bone particle and mixture for implantation, which can be bypacking, injection, scaffolding or any other suitable means, into apatient in a fracture healing procedure, by way of example.

Other ranges of bone particle sized and mixture can be employeddepending on the application which, in this example, was boneregeneration. Lower volumes and concentrations may be more suited forless intrusive bone repairs or more if larger if larger amounts ofmaterial are needed as in a hip defect or repair.

Variations in the present invention are possible in light of thedescription of it provided herein. While certain representativeembodiments and details have been shown for the purpose of illustratingthe subject invention, it will be apparent to those skilled in this artthat various changes and modifications can be made therein withoutdeparting from the scope of the subject invention. It is, therefore, tobe understood that changes can be made in the particular embodimentsdescribed, which will be within the full intended scope of the inventionas defined by the following appended claims.

What is claimed is:
 1. A biological composition comprising: a mixture ofmechanically selected allogeneic biologic material derived from bonemarrow having non-whole cellular components including vesicularcomponents and active and inactive components of biological activity,cell fragments, cellular excretions, cellular derivatives, andextracellular components; and wherein the mixture is compatible withbiologic function.
 2. The biological composition of claim 1 furthercomprises bone particles, the bone particles being added to the mixturederived from bone marrow.
 3. The biological composition of claim 2wherein the bone particles include a mixture of cortical bone particlesand cancellous bone particles.
 4. The biological composition of claim 1wherein the mixture of mechanically selected material derived from bonemarrow further includes a select number of non-whole cell fractionsincluding one or more of exosomes, transcriptosomes, proteasomes,membrane rafts, lipid rafts.
 5. The biological composition of claim 4wherein the combination of non-whole cell components with a selectnumber of the non-whole cell fractions sustains pluripotency in bothgraft or host cells or combinations thereof.
 6. The biologicalcomposition of claim 5 wherein the select number of the non-whole cellfractions sustains pluripotency in graft or host cells or combinationsthereof includes differentiated committed cells and non-differentiatedand non-committed cells.
 7. The biological composition of claim 2wherein the biological composition is predisposed to demonstrate orsupport elaboration of active volume or spatial geometry consistent inmorphology with that of endogenous bone.
 8. The biological compositionof claim 1 wherein the biological composition extends regenerativeresonance that compliments or mimics tissue complexity.
 9. Thebiological composition of claim 1 wherein the mixture is treated in aprotectant or cryoprotectant prior to preservation or cryopreservationor freeze drying.
 10. The biological composition of claim 9 wherein theprotectant or cryoprotectant creates a physical or electrical orchemical gradient or combination thereof for tissue regeneration. 11.The biological composition of claim 10 wherein the gradient has aphysical characteristic of modulus or topography such as charge density,field shape or cryo or chemo toxics tendencies.
 12. The biologicalcomposition of claim 10 wherein the gradient has a chemicalcharacteristic of spatially changing compositions of density or speciesof functional molecules, wherein the molecules can offer a fixedcatalytic function as a co-factor.
 13. The biological composition ofclaim 10 wherein the gradient has an electrical characteristic of chargebased or pH based or electron affinities that confer metastability inbiologic potential.
 14. The biological composition of claim 4 whereinthe bone marrow mixture which is derived from a cadaver hasseparation-enhanced non-whole cell fractions vitality including one ormore of the following: separating the fractions from cells heightenstheir vitality, reversing “arrest” of donors, responsive molecularcoupling, matrix quest in neutralizing inflammation or satience bybalancing stimulus for repair.
 15. The biological composition of claim 9wherein the protectant or cryoprotectant is a polyampholyte.
 16. Thebiological composition of claim 8 wherein the regenerative resonanceoccurs in the presence or absence of a refractory response.
 17. Thebiological composition of claim 9 wherein the cryopreservation occurs ata temperature that is sub-freezing.
 18. The biological composition ofclaim 17 wherein the cryopreservation temperature is from 0 degrees C.to −200 degrees C.
 19. The biological composition of claim 1 wherein themixture creates a physical or electrical or chemical gradient orcombination thereof for tissue regeneration.
 20. The biologicalcomposition of claim 19 wherein the gradient has a physicalcharacteristic such as modulus or topography.
 21. The biologicalcomposition of claim 19 wherein the gradient has a chemicalcharacteristic such as spatially changing compositions of density orspecies of functional molecules.
 22. The biological composition of claim19 wherein the gradient has an electrical characteristic such as chargebased or pH based.
 23. The biological composition of claim 1 can beorganelle fragments.
 24. The biological composition of claim 1 whereinactive an inactive components of biological activity can be extants ofthe human metabolome.
 25. The biological composition of claim 9 whereinthe composition is maintained at ambient temperature prior to freezedrying.